{"files"=>["https://ndownloader.figshare.com/files/1410690", "https://ndownloader.figshare.com/files/1410691", "https://ndownloader.figshare.com/files/1410692", "https://ndownloader.figshare.com/files/1410693", "https://ndownloader.figshare.com/files/1410694", "https://ndownloader.figshare.com/files/1410695", "https://ndownloader.figshare.com/files/1410696", "https://ndownloader.figshare.com/files/1410697", "https://ndownloader.figshare.com/files/1410698", "https://ndownloader.figshare.com/files/1410699", "https://ndownloader.figshare.com/files/1410700", "https://ndownloader.figshare.com/files/1410701"], "description"=>"<div><p>Reassortment of influenza viral RNA (vRNA) segments in co-infected cells can lead to the emergence of viruses with pandemic potential. Replication of influenza vRNA occurs in the nucleus of infected cells, while progeny virions bud from the plasma membrane. However, the intracellular mechanics of vRNA assembly into progeny virions is not well understood. Here we used recent advances in microscopy to explore vRNA assembly and transport during a productive infection. We visualized four distinct vRNA segments within a single cell using fluorescent <i>in situ</i> hybridization (FISH) and observed that foci containing more than one vRNA segment were found at the external nuclear periphery, suggesting that vRNA segments are not exported to the cytoplasm individually. Although many cytoplasmic foci contain multiple vRNA segments, not all vRNA species are present in every focus, indicating that assembly of all eight vRNA segments does not occur prior to export from the nucleus. To extend the observations made in fixed cells, we used a virus that encodes GFP fused to the viral polymerase acidic (PA) protein (WSN PA-GFP) to explore the dynamics of vRNA assembly in live cells during a productive infection. Since WSN PA-GFP colocalizes with viral nucleoprotein and influenza vRNA segments, we used it as a surrogate for visualizing vRNA transport in 3D and at high speed by inverted selective-plane illumination microscopy. We observed cytoplasmic PA-GFP foci colocalizing and traveling together en route to the plasma membrane. Our data strongly support a model in which vRNA segments are exported from the nucleus as complexes that assemble en route to the plasma membrane through dynamic colocalization events in the cytoplasm.</p></div>", "links"=>[], "tags"=>["Infectious diseases", "Viral diseases", "influenza", "intermediates", "fuse"], "article_id"=>954398, "categories"=>["Medicine"], "users"=>["Seema S. Lakdawala", "Yicong Wu", "Peter Wawrzusin", "Juraj Kabat", "Andrew J. Broadbent", "Elaine W. Lamirande", "Ervin Fodor", "Nihal Altan-Bonnet", "Hari Shroff", "Kanta Subbarao"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1003971.s001", "https://dx.doi.org/10.1371/journal.ppat.1003971.s002", "https://dx.doi.org/10.1371/journal.ppat.1003971.s003", "https://dx.doi.org/10.1371/journal.ppat.1003971.s004", "https://dx.doi.org/10.1371/journal.ppat.1003971.s005", "https://dx.doi.org/10.1371/journal.ppat.1003971.s006", "https://dx.doi.org/10.1371/journal.ppat.1003971.s007", "https://dx.doi.org/10.1371/journal.ppat.1003971.s008", "https://dx.doi.org/10.1371/journal.ppat.1003971.s009", "https://dx.doi.org/10.1371/journal.ppat.1003971.s010", "https://dx.doi.org/10.1371/journal.ppat.1003971.s011", "https://dx.doi.org/10.1371/journal.ppat.1003971.s012"], "stats"=>{"downloads"=>13, "page_views"=>21, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Influenza_A_Virus_Assembly_Intermediates_Fuse_in_the_Cytoplasm_/954398", "title"=>"Influenza A Virus Assembly Intermediates Fuse in the Cytoplasm", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2014-03-06 04:10:56"}

{"files"=>["https://ndownloader.figshare.com/files/1410653"], "description"=>"<p>Maximum intensity projection from inverted selective-plane illumination microscopy (iSPIM) movie of MDCK cells infected with WSN PA-GFP (MOI = 5) at 16 hpi, 36 seconds (s) after onset of tracking (A). The red-boxed region is highlighted in (B and C), and the red star indicates the region where colocalization of two GFP foci (marked in red and blue arrows) occurred. A single WSN PA-GFP focus trajectory; each red dot is a spot tracked in time, the yellow line is the overall trace, and white arrows indicate the direction of the PA-GFP movement (B). The total track duration is 46 s. Part C contains still frames of the red-boxed region in part A from 26–44 s, and illustrates colocalization and subsequent fusion of two foci. The red arrow indicates the PA-GFP focus that is being tracked and the blue arrow indicates the PA-GFP focus with which it fuses. All scale bars are 2 µm. An intensity versus depth plot of the colocalized foci, indicated by the red star in part A, demonstrates a single peak indicating that the fusion occurs in a single diffraction-limited spot (D). An intensity vs. time plot of the fused focus compared to an adjacent stationary focus, identified by the white circle in part C, during the same time frame (E). The asterisk marks the time corresponding to the colocalization event.</p>", "links"=>[], "tags"=>["Infectious diseases", "Viral diseases", "influenza", "wsn", "pa-gfp", "cytoplasm", "mdck"], "article_id"=>954367, "categories"=>["Medicine"], "users"=>["Seema S. Lakdawala", "Yicong Wu", "Peter Wawrzusin", "Juraj Kabat", "Andrew J. Broadbent", "Elaine W. Lamirande", "Ervin Fodor", "Nihal Altan-Bonnet", "Hari Shroff", "Kanta Subbarao"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.1003971.g006", "stats"=>{"downloads"=>0, "page_views"=>16, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Fusion_event_of_an_WSN_PA_GFP_focus_in_the_cytoplasm_of_MDCK_cells_/954367", "title"=>"Fusion event of an WSN PA-GFP focus in the cytoplasm of MDCK cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-03-06 04:10:56"}